Title: Visualization of the complex of the E coli ribosomal 50S subunit with tRNA and HSP15

Abstract:

Hsp15 is a heatshock protein that can rescue stalled 50S ribosomal particles. When thermal stress dissociates the 50S and 30S particles of translating ribosomes, a 50S particle with a tRNA carrying a nascent chain can result. Because the nascent chain is threaded through the 50S particle, the tRNA does not readily dissociate, and hence the 50S particle is rendered useless. However, opun binding of the Hsp15 to such stalled 50S particles, the tRNA can dissociate, and the 50S subunit has regained its translational activity. In order to investigate this process in molecular detail, we determined the structure of the complex of the 50S particle, carrying a tRNA with a nascent chain, and the Hsp15 to a resolution of about 1.2 nm. For this purpose we used the programs Cyclops (for particle picking), Eman (form 3D reconstruction) and CNS (for rigid body fitting of high-resolution X-ray structures of components into the low resolution EM density). One of the striking features of our structure determination is that the tRNA retracts its CCA-aminoacyl 3'-end from the peptidyl transferase centre and is clearly in a different orientation compared the 70S translating ribosome.